ABSTRACT Over the last two decades, our efforts to develop a syphilis vaccine have allowed identification of two antigens that confer high but not complete protection to syphilis in immunization/challenge experiments in the rabbit model. These two vaccine candidates were derived from conserved regions of selected members of the Treponema pallidum repeat (Tpr) antigens TprC/D2 and TprK. Immunity to these antigens has been shown to enhance pathogen clearance by opsonophagocytosis and provide substantial protection from chancre development at challenge sites compared to controls. In these early vaccine designs, however, several predicted surface epitopes of the TprC and TprD2 proteins were omitted because they showed a limited degree of antigenic variation, even though we demonstrated that they could provide additional targets for pathogen opsonization and phagocytosis by activated macrophages if used in combination with adjuvants inducing a Th1-type response. To attain a fully protective syphilis vaccine we aim to modify our early vaccine design to include additional protective epitopes of the TprC and TprD2 proteins. First (Aim 1), we will evaluate the protective activity of full-length recombinant TprC and TprD2 using a cocktail of antigens designed to cover all antigenic variants of these proteins, which will also contain the TprK conserved fragment. In parallel, we will conduct a series of experiments to map T-cell epitopes and protective B-cell epitopes of TprC and TprD2 to inform development of alternative platforms for antigen delivery, with the goal of delivering selected epitopes rather than on full length antigens. For these mapping experiments (also planned in Aim 1), we will use TprK as a control antigen, as B-and T-cell epitopes were already identified for this protein in the past. In lieu of recombinant proteins, we will engineer and test chimeric concatemers (Aim 2) and chimeric HPV-based viral-like particles (VLPs, Aim 3). These alternative delivery platforms will specifically induce immunity to targets of opsonizing antibodies on the treponemal surface, and likely enhance pathogen clearance upon infectious challenge. In Aim 4, we will combine our results and those obtained by Dr. Cameron (Project 1) with her selected vaccine candidate (Tp0751) to combine our most protective vaccine designs and delivery platform. Subsequently, a final immunization/challenge experiment with our fila vaccine design will be performed to assess overall level of protection and durability of immunity using a modern syphilis isolate to challenge immunized animals following rabbit immunization.